For as long as man has shared the planet with microorganisms there have been widespread outbreaks of infectious disease and subsequent widespread mortality associated with it. Although microorganisms and man frequently share a symbiotic relationship, microorganisms can, under some conditions, lead to sickness and death. The discovery, wide use and dissemination of antibiotics to treat microbial infection in both human and animal populations over the last one hundred or so years has done much to control, and in some instances, eradicate some microbes and associated infectious disease. However, microbes have a strong propensity to evolve and alter their genetic makeup when confronted with toxic substances that place them under life and death selective pressures. Therefore, emerging infectious diseases currently pose an important public health problem in both developed as well as developing countries. Not only have microbes evolved to evade and defeat current antibiotic therapeutics, but also there are novel and previously unrecognized and/or characterized bacterial, fungal, viral, and parasitic diseases that have emerged within the past two decades. Sass, Curr . Opin. in Drug Discov. & Develop. 2000, 3(5):646-654.
Since the accidental discovery of a penicillin-producing mold by Fleming there has been steady progress in synthesizing, isolating and characterizing new and more effective beta-lactam antibiotics. In addition to the great success of the beta-lactam family of antibiotics, the newer fluoroquinolones have a broad-spectrum of bactericidal activity as well as excellent oral bio-availability, tissue penetration and favorable safety and tolerability profiles. King et al., Am. Fam. Physician, 2000, 61, 2741-2748. A newly devised four-generation classification of the quinolone drugs accounts for the expanded antimicrobial spectrum of the more recently introduced fluoroquinolones and their clinical indications. The so-called first generation drugs, which include nalidixic acid, are capable of achieving minimal serum levels. The second-generation quinolones, such as ciprofloxacin, have an increased gram-negative and systemic activity. The third-generation drugs comprise pharmaceuticals such as levofloxacin and are have significant and expanded action against gram-positive bacteria and atypical pathogens. Finally, the fourth-generation quinolone drugs, which, to date, only includes trovofloxacin, are highly active against anaerobes in addition to the activity described for the third-generation drugs. Furthermore, the quinolone class of anti-microbial drugs can be divided based on their pharmacokinetic properties and bioavailability.
Mammalian epithelial surfaces are remarkable for their ability to provide critical physiologic functions in the face of frequent microbial challenges. The fact that these mucosal surfaces remain infection-free in the normal host suggests that highly effective mechanisms of host defense have evolved to protect these environmentally exposed tissues. Throughout the animal and plant kingdoms, endogenous genetically encoded antimicrobial peptides have been shown to be key elements in the response to epithelial compromise and microbial invasion. Zasloff, Curr. Opin. Immunol., 1992, 4, 3-7; and Bevins, Ciba Found. Symp., 1994, 186, 250-69. In mammals, a variety of such peptides have been identified, including the well-characterized defensins and cathelicidins and others (andropin, magainin, tracheal antimicrobial peptide, and PR-39; see Bevins, Ciba Found. Symp., 1994, 186, 250-69 and references therein). A major source of these host defense molecules is circulating phagocytic leukocytes. However, more recently, it has been shown that resident epithelial cells of the skin and respiratory, alimentary, and genitourinary tracts also synthesize and release antimicrobial peptides. Both in vitro and in vivo data support the hypothesis that these molecules are important contributors to intrinsic mucosal immunity. Alterations in their level of expression or biologic activity can predispose the organism to microbial infection. Huttner et al., Pediatr. Res., 1999, 45, 785-94.
Across the evolutionary scale species from insects to mammals to plants defend themselves against invading pathogenic microorganisms by utilizing cationic antimicrobial peptides that rapidly kill microbes without exerting toxicity to the host. Physicochemical peptide-lipid interactions provide attractive mechanisms for innate immunity as discussed below. Many of these peptides form cationic amphipathic secondary structures, typically alpha-helices and beta-sheets, which can selectively interact with anionic bacterial membranes via electrostatic interactions. Rapid, peptide-induced membrane permeabilization and subsequent cellular lysis is the result. Matsuzaki, Biochim. Biophys. Acta, 1999, 1462, 1-10.
The primary structures of a large number of these host-defense peptides have been determined. While there is no primary structure homology, the peptides are characterized by a preponderance of cationic and hydrophobic amino acids. The secondary structures of many of the host-defense peptides have been determined by a variety of techniques. Sitaram et al., Biochim, Biophys. Acta, 1999, 1462, 29-54. The acyclic peptides tend to adopt helical conformation, especially in media of low dielectric constant, whereas peptides with more than one disulfide bridge adopt beta-structures.
As described above, one reason for the rise in microbial drug resistance to the first line antimicrobial therapies in standard use today is the inappropriate and over-use of prescription antibiotics. Although bacteria are the most common organisms to develop drug-resistance, there are numerous examples of demonstrated resistance in fungi, viruses, and parasites. The development of a resistant phenotype is a complex phenomenon that involves an interaction of the microorganism, the environment, and the patient, separately as well as in combination. Sitaram et al., Biochim. Biophys. Acta, 1999, 1462, 29-54. The microorganism in question may develop resistance while under antibiotic selection or it may be a characteristic of the microbe prior to exposure to a given agent. There are a number of mechanisms of resistance to antibiotics that have been described, including genes that encode antibiotic resistance enzymes that are harbored on extra-chromosomal plasmids as well as DNA elements (e.g. transposable elements) that can reside either extra-chromosomally or within the host genome.
Due to the ability of microorganisms to acquire the ability to develop resistance to antibiotics there is a need to continually develop novel antibiotics. Traditional methods to develop novel antibiotics have included medicinal chemistry approaches to modify existing antibiotics (Kang et al., Bioorg. Med. Chem. Lett., 2000, 10, 95-99) as well as isolation of antibiotics from new organisms (Alderson et al., Res. Microbiol., 1993, 144, 665-72). Each of these methods, however, has limitations. The traditional medicinal chemistry approach entails modification of an existing molecule to impart a more effective activity. The chemist makes a “best guess” as to which parts of the molecule to alter, must then devise a synthetic strategy, synthesize the molecule, and then have it tested. This approach is laborious, requires large numbers of medicinal chemists and frequently results in a molecule that is lower in activity than the original antibiotic. The second approach, isolation of novel antimicrobial agents, requires screening large numbers of diverse organisms for novel antimicrobial activity. Then, the activity must be isolated from the microorganism. This is not a small task, and frequently takes many years of hard work to isolate the active molecule. Even after the molecule is identified, it may not be possible for medicinal chemists to effectively devise a synthetic strategy due to the complexity of the molecule. Furthermore, the synthetic strategy must allow for a cost-effective synthesis. Therefore, a method that would allow for creation of more effective antibiotics from existing molecules or allow rapid isolation of novel antimicrobial agents is needed to combat the ever-growing list of antibiotic resistant organisms. The present invention described herein is directed to the use of random genetic mutation of a cell to produce novel antibiotics by blocking the endogenous mismatch repair (MMR) activity of a host cell. The cell can be a mammalian cell that produces an antimicrobial agent naturally, or a cell that is placed under selective pressure to obtain a novel antimicrobial molecule that attacks a specific microbe. Moreover, the invention describes methods for obtaining enhanced antimicrobial activity of a cell line that produces an antimicrobial activity due to recombinant expression or as part of the innate capacity of the cell to harbor such activity.
In addition, the generation of genetically altered host cells that are capable of secreting an antimicrobial activity, which can be protein or non-protein based, will be valuable reagents for manufacturing the entity for clinical studies. An embodiment of the invention described herein is directed to the creation of genetically altered host cells with novel and/or increased antimicrobial production that are generated by a method that interferes with the highly ubiquitous and phylogenetically conserved process of mismatch repair.
The present invention facilitates the generation of novel antimicrobial agents and the production of cell lines that express elevated levels of antimicrobial activity. Advantages of the present invention are further described in the examples and figures described herein.